Ligation of the T-cell receptor initiates a cascade of intracellular signaling events resulting in the proliferation and differentiation of the activated cell. Many of the phenotypic changes which define T-cell activation result from new gene transcription (Ullman, et al., Annu. Rev. Immunol. 8:421-452 (1990)). Activation-induced changes in cell surface proteins resulting from a primary stimulus play a particularly important role in regulating downstream proliferative and differentiative responses. Events mediated at the cell surface include binding of soluble factors and interactions with other cells and extracellular matrix. In vivo, activated T cells play an instrumental role in the propagation of immunologically mediated inflammation (Brezinschek, et al., J. Immunol., 154:3062-77 (1995)).
The development and progression of inflammation is dependent upon the infiltration of leukocytes into the affected tissues. The accumulation of leukocytes into tissues involves receptor-mediated interactions with the endothelial cell lining of postcapillary venules, extravasation, and migration toward and localization within the inflammatory site (Shimizu, et al., FASEB J. 5:2292-2299 (1992)). A large body of work has shown that the combinatorial use of multiple adhesion and chemoattractant receptors appears to regulate selection of subclasses of leukocytes emigrating at inflammatory sites as well as the distinctive recirculation behavior of lymphocyte subsets (Springer, Cell 76:301-314 (1994)). Little is known about the range of receptor-ligand interactions in leukocytes that regulate their localization within the tissue microenvironment following extravasation.
Once at the site of inflammation, immune cells undergo additional phenotypic changes that contribute to eliminating the foreign antigen and to amplifying the inflammatory response. Various soluble mediators of inflammation such as prostaglandins, leukotrienes, complement fragments, platelet-activating factors, chemokines, and formyl peptides, among others (Murphy, Annu. Rev. Immunol. 12:593-633 (1994)) bind to specific receptors that are part of a very large and diverse class of receptors that span the membrane seven times (7TM receptors). 7TM receptors, also called G protein coupled receptors, transduce signals following ligand binding via their association with heterotrimeric G proteins (Martens, PROGRESS IN BRAIN RESEARCH, Joose, et al., (eds.), pp. 201-214 (1992)). Receptor coupled G protein activation in turn regulates a variety of enzymes (such as adenyl cyclase, phospholipase Cb, phosphoinositide 3-kinase), ion channels and transporters (Neer, Cell 80:249-257 (1995)).
The family of 7TM receptors is probably the largest receptor family known, a with hundreds of receptors cloned to date. The receptors bind a wide structural array of ligands including various types of hormones, neurotransmitters, lipids, peptides, and odorants (Spiegel, G PROTEINS, Spiegel., et al., (eds.), R. G. Landes Co., Austin. pp. 6-17 (1994)). The defining feature and the areas of greatest homology among the 7TM receptors are in the seven transmembrane regions (Probst, et al., DNA and Cell Biol. 11:1-20 (1992)). Some residues are found in virtually all 7TM receptors and may mediate evolutionarily-conserved tertiary structural requirements for functional activity. Other residues are conserved among subfamilies that bind similar ligands and have been shown to contribute to ligand binding and/or specificity (Savarese and Fraser, Biochem. J. 283:1-19 (1992)). In the case of the glucagon/calcitonin receptor subfamily, relatedness based on sequence identity is apparent despite the diversity of the peptides that bind to these receptors (Attwood and Findlay, Protein Eng. 7:195-203 (1994)).
The structural features required for ligand binding and receptor activation have been investigated and found to vary according to ligand and receptor subfamily (Coughlin, Curr. Opin. in Cell Biol. 6:191-197 (1994)). Many small ligands such as 11-cis-retinal, serotonin, and acetylcholine, bind within the cavity formed by the receptors"" transmembrane domains (Baldwin, Curr. Opin. in Cell Biol. 6:180-190 (1994); Dohlman, et al., Annu. Rev. Biochem. 283:1-19 (1992); and Savarese and Fraser, Biochem. J. 283:1-19 (1992)). Other ligands such as peptides and glycoprotein hormones require amino-terminal exodomains and most likely some portion of the extracellular loops for binding, but signaling requires the seven membrane spans (Holtmann, et al., J. Biol. Chem. 270:14394-14398 (1995) and Nagayama, et al., Proc. Nat""l Acad. Sci. (USA) 88:902-905 (1991)). A remarkable signaling mechanism has been described for the thrombin receptor in which thrombin cleaves its receptor""s amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor through interactions with the interhelical pocket (Vu, et al., Cell 64:1057-1068 (1991)).
Hamann, et al. (J. Immunol. 155:1942-1950 (1995)) report the isolation of a glycoprotein designated CD97. Seven hydrophobic segments within CD97 suggest that this glycoprotein is a 7TM molecule. CD97 is induced on the surface of most leukocytes upon activation. In its mature form, Hamann, et al. indicate that CD97 is a single chain glycoprotein of 722 amino acids in length with a molecular weight of 75 to 85 kDa.
The present invention relates to the previously unrecognized a subunit of CD97. The xcex1 subunit binds to the xcex2 subunit of CD97 to form an xcex1xcex2 heterodimer. The xcex1 subunit is localized extracellularly on T-cells. Upon activation, expression of the xcex1 subunit is dramatically increased and shed into the external medium. The xcex1 subunit plays a role in angiogenesis, inflammation, and atherosclerosis. Detection and inhibition of xcex1 subunit expression provides diagnostic and therapeutic methods for these disease states.
In one aspect, the present invention relates to an isolated protein comprising a soluble CD97 xcex1 subunit. The soluble CD97 xcex1 subunit is selected from the group consisting of xcex11, xcex12, and xcex13. The xcex11, xcex12 and xcex13 subunits are related in that they all originate as a proprotein with the xcex2 subunit (FIG. 1) and are processed in the endoplasmic reticulum or early golgi to a specific xcex1 subunit with an identifying number of EGF repeats and a non-covalently linked xcex2 subunit.
The xcex13 subunit has a molecular weight of about 45 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of EGF-1 (SEQ ID NO:1), EGF-2 (SEQ ID NO:2), and EGF-5 (SEQ ID NO:5), and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex12 subunit has a molecular weight of about 50 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex11 subunit has a molecular weight of about 55 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. In some embodiments the xcex11 subunit further comprises an EGF-like repeat selected from the group consisting of EGF-3 (SEQ ID NO:3), and EGF-4 (SEQ ID NO:4). In other embodiments the xcex12 subunit further comprises EGF-like repeat SEQ ID NO:3. Conveniently, the isolated protein is recombinantly produced.
In another aspect, the present invention relates to an isolated nucleic acid encoding a soluble CD97 xcex1 subunit protein. The CD97 xcex1 subunit protein is selected from the group consisting of xcex11, xcex12, and xcex13. The xcex13 subunit has a molecular weight of about 45 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex12 subunit has a molecular weight of about 50 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex11 subunit has a molecular weight of about 55 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6.
In some embodiments, the nucleic acid encodes a CD97 xcex1 subunit selected from the group consisting of xcex11 and xcex12, further comprising an EGF-like repeat selected from the group consisting of SEQ ID NO:3, and SEQ ID NO:4. In some embodiments the xcex12 subunit further comprises EGF-like repeat SEQ ID NO:3. In additional embodiments, the nucleic acid is operably linked in forward or reverse orientation to a promoter, either of which can be used to transfect a host cell.
In an additional aspect the present invention relates to an isolated mammalian protein comprising a soluble CD97 xcex1 subunit. The CD97 xcex1 subunit is an extracellular protein comprising at least 10 contiguous amino acids from the protein of SEQ ID NO:6, is increased at least five-fold upon maximal activation of a T-cell with a T-cell mitogen, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6.
In yet another aspect, the present invention relates to an isolated nucleic acid, encoding a soluble CD97 xcex1 subunit, of at least 25 nucleotides in length, wherein the CD97 xcex1 subunit is selected from the group consisting of xcex11 and xcex12. The xcex12 subunit has a molecular weight of about 50 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex11 subunit has a molecular weight of about 55 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. In this aspect, the nucleic acid specifically hybridizes, under stringent conditions, at least two-fold above background to a CD97 nucleic acid in a human genomic library.
In an additional aspect, the present invention relates to an antibody composition specifically reactive, under immunologically reactive conditions, to a soluble CD97 xcex1 subunit selected from the group consisting of xcex11 and xcex12. The xcex12 subunit has a molecular weight of about 50 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex11 subunit has a molecular weight of about 55 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. In some embodiments the antibody composition comprises at least three unique antibodies.
In a further aspect, the present invention relates to a method for determining the degree of inflammation at a site in a mammal. The method comprises the steps of contacting an antibody composition to a biological sample from the site, wherein the antibody composition is specifically reactive, under immunologically reactive conditions, to a soluble CD97 xcex1 subunit selected from the group consisting of xcex11, xcex12, and xcex13.
The xcex13 subunit has a molecular weight of about 45 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex12 subunit has a molecular weight of about 50 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The xcex11 subunit has a molecular weight of about 55 kDa in non-glycosylated form, has an EGF-like repeat selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5, and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6.
In the method, the antibody composition is incubated with the biological fluid. under immunologically reactive conditions conducive to formation of an specific antibody:CD97 xcex1 subunit complex, wherein detection of the amount of the complex indicates the extent of inflammation at the site. In preferred embodiments, the biological sample is selected from the group consisting of blood, synovial fluid, and cerebrospinal fluid.
In yet another aspect, the present invention relates to a method for inhibiting angiogenesis associated with chronic inflammation in a mammal, comprising administering a therapeutically effective amount of a CD97 antagonist selected from the group consisting of CD97 subunit antisense nucleic acid, CD97 subunit xcex1 decoy protein, and anti-CD97 xcex1 subunit antibody, wherein the CD97-subunit is selected from the group consisting of xcex11, xcex12, xcex13 and xcex2. Subunits xcex13, xcex12, and xcex11 are as provided supra. The xcex2 subunit has a molecular weight of about 28 kDa as an unglycosylated protein and is immunologically cross-reactive to an antibody that is specifically reactive to the protein of SEQ ID NO:6. The therapeutically effective amount is administered topically or parenterally.
In a further aspect, the present invention relates to a method of treating or inhibiting CD97 associated inflammation in a mammal, comprising administering a therapeutically effective amount of a CD97 antagonist selected from the group consisting of CD97 subunit antisense nucleic acid, CD97 subunit xcex1 decoy protein, and anti-CD97 subunit antibody, and wherein the CD97-subunit is selected from the group consisting of xcex11, xcex12, and xcex13. Subunits xcex13, xcex12, and xcex11 are as provided supra.
In yet another aspect, the present invention relates to a method for inhibiting atherosclerosis, comprising administering a therapeutically effective amount of a CD97 antagonist selected from the group consisting of CD97 subunit antisense nucleic acid, CD97 subunit xcex1 decoy protein, and anti-CD97 xcex1 subunit antibody, wherein the CD97-subunit is selected from the group consisting of xcex11, xcex12, xcex13 and xcex2. Subunits xcex13, xcex12, xcex11, and xcex2 are as provided supra. The therapeutically effective amount is administered topically or parenterally.
In a further aspect, the present invention relates to a method for identifying a compound which inhibits soluble CD97 xcex1 subunit expression. The method comprises contacting, under cell culture conditions, the compound with a resting T-cell and an effective amount of a T-cell mitogen. In the method the compound is present in at least nanomolar concentrations. Changes in the expression level of the CD97 xcex1 subunit are assayed for, wherein the subunit is selected from the group consisting of xcex11, xcex12, and xcex13. Subunits xcex13, xcex12, and xcex11 are as provided supra. A reduced level of expression of the subunit relative to a negative control identifies the compound as an inhibitor. In preferred embodiments, the T-cell mitogen is selected from the group consisting of phytohemagglutinin, concanavalin A, phorbol 12-myristate 13-acetate, and pokeweed mitogen. Typically, changes in the expression of the CD97 xcex1 subunit are determined by immunoassay or nucleic acid assay.